Capsular polysaccharide solubilisation and combination vaccines

ABSTRACT

Precipitated bacterial capsular polysaccharides can be efficiently re-solubilized using alcohols as solvents. The invention provides a process for purifying a bacterial capsular polysaccharide, comprising the steps of (a) precipitation of said polysaccharide, followed by (b) solubilization of the precipitated polysaccharide using ethanol. CTAB can be used for step (a). The material obtained, preferably following hydrolysis and sizing, can be conjugated to a carrier protein and formulated as a vaccine. Also, in vaccines comprising saccharides from both serogroups A and C, the invention provides that the ratio (w/w) of MenA saccharide:MenC saccharide is &gt;1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 10/481,457, which is the U.S. National Phase of InternationalApplication No. PCT/IB02/03191, filed Jun. 20, 2002 and published inEnglish, which claims priority to G.B. Application No. 0115176.0, filedJun. 20, 2001. The teachings of the above applications are incorporatedin their entirety by reference.

All documents cited herein are incorporated by reference in theirentirety.

TECHNICAL FIELD

This invention is in the field of vaccines, particularly againstmeningococcal infection and disease.

BACKGROUND ART

Neisseria meningitidis is a Gram negative human pathogen. It colonisesthe pharynx, causing meningitis and, occasionally, septicaemia in theabsence of meningitis. It is closely related to N. gonorrhoeae, althoughone feature that clearly differentiates meningococcus is the presence ofa polysaccharide capsule that is present in all pathogenic meningococci.

Based on the organism's capsular polysaccharide, twelve serogroups of N.meningitidis have been identified (A, B, C, H, I, K, L, 29E, W135, X, Yand Z). Group A is the pathogen most often implicated in epidemicdisease in sub-Saharan Africa. Serogroups B and C are responsible forthe vast majority of cases in USA and in most developed countries.Serogroups W135 and Y are responsible for the remaining cases in USA anddeveloped countries.

Capsular polysaccharides from N. meningitidis are typically prepared bya process comprising the steps of polysaccharide precipitation (e.g.using a cationic detergent), ethanol fractionation, cold phenolextraction (to remove protein) and ultracentrifugation (to remove LPS)[e.g. ref. 1].

A tetravalent vaccine of capsular polysaccharides from serogroups A, C,Y and W135 has been known for many years [2, 3] and has been licensedfor human use. Although effective in adolescents and adults, it inducesa poor immune response and short duration of protection and cannot beused in infants [e.g. 4]. This is because polysaccharides are Tcell-independent antigens that induce a weak immune response that cannotbe boosted. The polysaccharides in this vaccine are not conjugated andare present at a 1:1:1:1 ratio [5]. MENCEVAX ACWY™ contains 50 μg ofeach purified polysaccharide once reconstituted from its lyophilisedform.

Conjugated serogroup C oligosaccharides have also been approved forhuman use [e.g. MENJUGATE™; ref. 6]. There remains, however, a need forimprovements in conjugate vaccines against serogroups A, W135 and Y, andin their manufacture.

DISCLOSURE OF THE INVENTION

The invention provides a process for purifying a bacterial capsularpolysaccharide, comprising the steps of (a) precipitation of saidpolysaccharide, followed by (b) solubilisation of the precipitatedpolysaccharide using ethanol. The polysaccharide can be used to preparevaccines, such as conjugate vaccines, in particular against N.meningitidis serogroups A, W135 and Y.

Precipitation and Ethanol Solubilisation

Many techniques for precipitating soluble polysaccharides are known inthe art. Preferred methods use one or more cationic detergents. Thedetergents preferably have the following general formula:

wherein:

-   -   R₁, R₂ and R₃ are the same or different and each signifies alkyl        or aryl; or R₁ and R₂ together with the nitrogen atom to which        these are attached form a 5- or 6-membered saturated        heterocyclic ring, and R₃ signifies alkyl or aryl; or R₁, R₂ and        R₃ together with the nitrogen atom to which these are attached        form a 5- or 6-membered heterocyclic ring, unsaturated at the        nitrogen atom,    -   R₄ signifies alkyl or aryl, and    -   X⁻ signifies an anion.

Particularly preferred detergents for use in the method aretetrabutylammonium and cetyltrimethylammonium salts (e.g. the bromidesalts). Cetyltrimethylammonium bromide (‘CTAB’) is particularlypreferred [8]. CTAB is also known as hexadecyltrimethylammonium bromide,cetrimonium bromide, Cetavlon and Centimide. Other detergents includehexadimethrine bromide and myristyltrimethylammonium salts.

Capsular polysaccharides are released into media during culture.Accordingly, the starting material for precipitation will typically bethe supernatant from a centrifuged bacterial culture or will be aconcentrated culture.

The precipitation step may be selective for polysaccharides, but it willtypically also co-precipitate other components (e.g. proteins, nucleicacid etc.).

Precipitated polysaccharide may be collected by centrifugation prior tosolubilisation.

After precipitation, the polysaccharide (typically in the form of acomplex with the cationic detergent) is re-solubilised. It is preferredto use a solvent which is relatively selective for the polysaccharide inorder to minimise contaminants (e.g. proteins, nucleic acid etc.).Ethanol has been found to be advantageous in this respect, and it ishighly selective for the CTAB-polysaccharide complex. Other loweralcohols may be used (e.g. methanol, propan-1-ol, propan-2-ol,butan-1-ol, butan-2-ol, 2-methyl-propan-1-ol, 2-methyl-propan-2-ol,diols etc.)

The ethanol is preferably added to the precipitated polysaccharide togive a final ethanol concentration (based on total content of ethanoland water) of between 50% and 95% (e.g. around 55%, 60%, 65%, 70%, 75%,80%, 85%, or around 90%), and preferably between 75% and 95%. Theoptimum final ethanol concentration may depend on the serogroup of thebacterium from which the polysaccharide is obtained.

The ethanol may be added to the precipitated polysaccharide in pure formor may be added in a form diluted with a miscible solvent (e.g. water).Preferred solvent mixtures are ethanol:water mixtures, with a preferredratio of between around 70:30 and around 95:5 (e.g. 75:25, 80:20, 85:15,90:10).

Compared with conventional processes for preparing capsularpolysaccharides, the two-step process of precipitation followed byethanol extraction is quicker and simpler.

In contrast to the process described in ref. 9, the process usescationic detergent rather than anionic detergent. Unlike the process ofref. 10, the polysaccharide is re-solubilised using ethanol, rather thanby cation exchange using calcium or magnesium salts. Unlike the processof ref. 11, precipitation does not require an inert porous support.Furthermore, unlike prior art processes, alcohol is used tore-solubilise the polysaccharide rather than to precipitate it.

The bacterial capsular polysaccharide will usually be from Neisseria.Preferably it is from N. meningitidis, including serogroups A, B, C,W135 & Y. Preferred serogroups are A, W135 & Y.

The process is also suitable for preparing capsular polysaccharide fromHaemophilus influenzae (particularly type B, or ‘Hib’) and fromStreptococcus pneumoniae (pneumococcus).

Further Processing of the Solubilised Polysaccharide

After re-solubilisation, the polysaccharide may be further treated toremove contaminants. This is particularly important in situations whereeven minor contamination is not acceptable (e.g. for human vaccineproduction). This will typically involve one or more steps offiltration.

Depth filtration may be used. This is particularly useful forclarification.

Filtration through activated carbon may be used. This is useful forremoving pigments and trace organic compounds. It can be repeated until,for example, OD_(275nm)<0.2.

Size filtration or ultrafiltration may be used.

Once filtered to remove contaminants, the polysaccharide may beprecipitated for further treatment and/or processing. This can beconveniently achieved by exchanging cations (e.g. by the addition ofcalcium or sodium salts).

The polysaccharide may be chemically modified. For instance, it may bemodified to replace one or more hydroxyl groups with blocking groups.This is particularly useful for MenA [12]. Polysaccharides fromserogroup B may be N-propionylated [13].

The (optionally modified) polysaccharide will typically be hydrolysed toform oligosaccharides. This is preferably performed to give a finalaverage degree of polymerisation (DP) in the oligosaccharide of lessthan 30 (e.g. between 10 and 20, preferably around 10 for serogroup A;between 15 and 25 for serogroups W135 and Y, preferably around 15-20;etc.). Oligosaccharides are preferred to polysaccharides for use invaccines. DP can conveniently be measured by ion exchange chromatographyor by calorimetric assays [14].

If hydrolysis is performed, the hydrolysate will generally be sized inorder to remove short-length oligosaccharides. This can be achieved invarious ways, such as ultrafiltration followed by ion-exchangechromatography. Oligosaccharides with a degree of polymerisation of lessthan or equal to about 6 are preferably removed for serogroup A, andthose less than around 4 are preferably removed for serogroups W135 andY.

To enhance immunogenicity, polysaccharides or oligosaccharides of theinvention are preferably conjugated to a carrier (FIG. 18). Conjugationto carrier proteins is particularly useful for paediatric vaccines [e.g.ref. 15] and is a well known technique [e.g. reviewed in refs. 16 to 24,etc.].

Preferred carrier proteins are bacterial toxins or toxoids, such asdiphtheria or tetanus toxoids. The CRM₁₉₇ diphtheria toxoid [25, 26, 27]is particularly preferred. Other suitable carrier proteins include theN. meningitidis outer membrane protein [28], synthetic peptides [29,30], heat shock proteins [31, 32], pertussis proteins [33, 34],cytokines [35], lymphokines [35], hormones [35], growth factors [35],artificial proteins comprising multiple human CD4⁺ T cell epitopes fromvarious pathogen-derived antigens [36, protein D from H. influenzae[37], toxin A or B from C. difficile [38], etc. It is possible to usemixtures of carrier proteins.

Conjugates with a saccharide:protein ratio (w/w) of between 0.5:1 (i.e.excess protein) and 5:1 (i.e. excess saccharide) are preferred, andthose with a ratio between 1:1.25 and 1:2.5 are more preferred.

A single carrier protein may carry multiple different saccharides [39].Conjugates may be used in conjunction with free carrier protein [40].

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

The saccharide will typically be activated or functionalised prior toconjugation. Activation may involve, for example, cyanylating reagentssuch as CDAP (e.g. 1-cyano-4-dimethylamino pyridinium tetrafluoroborate[41, 42, etc.]). Other suitable techniques use carbodiimides,hydrazides, active esters, norborane, p-nitrobenzoic acid,N-hydroxysuccinimide, S—NHS, EDC, TSTU; see also the introduction toreference 22).

Linkages via a linker group may be made using any known procedure, forexample, the procedures described in references 43 and 44. One type oflinkage involves reductive amination of the polysaccharide, coupling theresulting amino group with one end of an adipic acid linker group, andthen coupling a protein to the other end of the adipic acid linker group[20, 45, 46]. Other linkers include B-propionamido [47],nitrophenyl-ethylamine [48], haloacyl halides [49], glycosidic linkages[50], 6-aminocaproic acid [51], ADH [52], C₄ to C₁₂ moieties [53] etc.As an alternative to using a linker, direct linkage can be used. Directlinkages to the protein may comprise oxidation of the polysaccharidefollowed by reductive amination with the protein, as described in, forexample, references 54 and 55.

A process involving the introduction of amino groups into the saccharide(e.g. by replacing terminal ═O groups with —NH₂) followed byderivatisation with an adipic diester (e.g. adipic acidN-hydroxysuccinimido diester) and reaction with carrier protein ispreferred.

After conjugation, free and conjugated saccharides can be separated.There are many suitable methods, including hydrophobic chromatography,tangential ultrafiltration, diafiltration etc. [see also refs. 56 & 57,etc.].

Mixtures and Compositions Comprising the Saccharides

The oligosaccharides, polysaccharides and conjugates of the inventionmay mixed with other biological molecules. Mixtures of saccharides frommore than one serogroup of N. meningitidis are preferred e.g.compositions comprising saccharides from serogroups A+C, A+W135, A+Y,C+W135, C+Y, W135+Y, A+C+W135, A+C+Y, C+W135+Y, A+C+W135+Y, etc. It ispreferred that the protective efficacy of individual saccharide antigensis not removed by combining them, although actual immunogenicity (e.g.ELISA titres) may be reduced.

Where a saccharide from serogroup C is used, this preferably has from˜12 to ˜22 repeating units.

Saccharides from different serogroups of N. meningitidis may beconjugated to the same or different carrier proteins.

Where a mixture comprises capsular saccharides from both serogroups Aand C, it is preferred that the ratio (w/w) of MenA saccharide:MenCsaccharide is greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher).Surprisingly, improved immunogenicity of the MenA component has beenobserved when it is present in excess (mass/dose) to the MenC component.

Where a mixture comprises capsular saccharides (e.g. oligosaccharides)from serogroup W135 and at least one of serogroups A, C and Y, it hassurprisingly been found that the immunogenicity of the MenW135saccharide is greater when administered in combination with thesaccharide(s) from the other serogroup(s) than when administered alone(at the same dosage etc.) [cf. ref. 58]. Thus the capacity of theMenW135 antigen to elicit an immune response is greater than the immuneresponse elicited by an equivalent amount of the same antigen whendelivered without association with the antigens from the otherserogroups. Such enhanced immunogenicity can be determined byadministering the MenW135 antigen to control animals and the mixture totest animals and comparing antibody titres against the two usingstandard assays such as bactericidal titres, radioimmunoassay and ELISAsetc. Vaccines comprising synergistic combinations of saccharides fromserogroup W135 and other serogroups are immunologically advantageous.They allow enhanced anti-W135 responses and/or lower W135 doses.

Where a mixture comprises capsular saccharides from serogroup Y and oneor both of serogroups C and W135, it is preferred that the ratio (w/w)of MenY saccharide:MenW135 saccharide is greater than 1 (e.g. 2:1, 3:1,4:1, 5:1, 10:1 or higher) and/or that the ratio (w/w) of MenYsaccharide:MenC saccharide is less than 1 (e.g. 1:2, 1:3, 1:4, 1:5, orlower).

Preferred ratios (w/w) for saccharides from serogroups A:C:W135:Y are:1:1:1:1; 1:1:1:2; 2:1:1:1; 4:2:1:1; 8:4:2:1; 4:2:1:2; 8:4:1:2; 4:2:2:1;2:2:1:1; 4:4:2:1; 2:2:1:2; 4:4:1:2; and 2:2:2:1.

The mixtures may also comprise proteins. It is preferred to includeproteins from serogroup B of N. meningitidis [e.g. refs. 59 to 64] orOMV preparations [e.g. refs. 65 to 68 etc.].

Non-meningococcal and non-neisserial antigens, preferably ones that donot diminish the immune response against the meningococcal components,may also be included. Ref. 69, for instance, discloses combinations ofoligosaccharides from N. meningitidis serogroups B and C together withthe Hib saccharide. Antigens from pneumococcus, hepatitis A virus,hepatitis B virus, B. pertussis, diphtheria, tetanus, Helicobacterpylori, polio and/or H. influenzae are preferred. Particularly preferrednon-neisserial antigens include:

-   -   antigens from Helicobacter pylori such as CagA [70 to 73], VacA        [74, 75], NAP [76, 77, 78], HopX [e.g. 79], HopY [e.g. 79]        and/or urease.    -   a saccharide antigen from Streptococcus pneumoniae [e.g. 80, 81,        82].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 83, 84].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 84, 85], with surface antigen preferably        being adsorbed onto an aluminium phosphate [86].    -   a saccharide antigen from Haemophilus influenzae B [e.g. 87],        preferably non-adsorbed or adsorbed onto an aluminium phosphate        [88].    -   an antigen from hepatitis C virus [e.g. 89].    -   an antigen from N. gonorrhoeae [e.g. 59 to 62].    -   an antigen from Chlamydia pneumoniae [e.g. refs. 90 to 96].    -   an antigen from Chlamydia trachomatis [e.g. 97].    -   an antigen from Porphyromonas gingivalis [e.g. 98].    -   polio antigen(s) [e.g. 99, 100] such as IPV.    -   rabies antigen(s) [e.g. 101] such as lyophilised inactivated        virus [e.g. 102, RabAvert™].    -   measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11        of ref. 103].    -   influenza antigen(s) [e.g. chapter 19 of ref. 103], such as the        haemagglutinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis [e.g. 104].    -   an antigen from Streptococcus agalactiae (group B streptococcus)        [e.g. 105, 106].    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. 106, 107, 108].    -   an antigen from Staphylococcus aureus [e.g. 109].    -   antigen(s) from a paramyxovirus such as respiratory syncytial        virus (RSV [110, 111]) and/or parainfluenza virus (PIV3 [112]).    -   an antigen from Bacillus anthracis [e.g. 113, 114, 115].    -   an antigen from a virus in the flaviviridae family (genus        flavivirus), such as from yellow fever virus, Japanese        encephalitis virus, four serotypes of Dengue viruses, tick-borne        encephalitis virus, West Nile virus.    -   a pestivirus antigen, such as from classical porcine fever        virus, bovine viral diarrhoea virus, and/or border disease        virus.    -   a parvovirus antigen e.g. from parvovirus B 19.    -   a tetanus toxoid [e.g. ref. 116].    -   pertussis holotoxin (PT) and filamentous haemagglutinin (FHA)        from B. pertussis, optionally also in combination with pertactin        and/or agglutinogens 2 and 3 [e.g. refs. 117 & 118].    -   cellular pertussis antigen.

The mixture may comprise one or more of these further antigens, whichmay be detoxified where necessary (e.g. detoxification of pertussistoxin by chemical and/or genetic means).

Where a diphtheria antigen is included in the mixture it is preferredalso to include tetanus antigen and pertussis antigens. Similarly, wherea tetanus antigen is included it is preferred also to include diphtheriaand pertussis antigens. Similarly, where a pertussis antigen is includedit is preferred also to include diphtheria and tetanus antigens.

Antigens in the mixture will typically be present at a concentration ofat least 1 μg/ml each In general, the concentration of any given antigenwill be sufficient to elicit an immune response against that antigen.

As an alternative to using proteins antigens in the mixture, nucleicacid encoding the antigen may be used. Protein components of the mixturemay thus be replaced by nucleic acid (preferably DNA e.g. in the form ofa plasmid) that encodes the protein.

Multivalent Saccharide Vaccines

The invention also provides vaccines and immunogenic compositionscomprising capsular saccharides from at least two (i.e. 2, 3 or 4) ofserogroups A, C, W135 and Y of N. meningitidis, wherein said capsularsaccharides are conjugated to carrier protein(s) and/or areoligosaccharides. Where the vaccine has only two conjugatedoligosaccharides or polysaccharides from serogroups A, C, W135 and Y,these are preferably not from serogroups A and C (cf. refs. 6, 119 &120). Preferred compositions comprise saccharides from serogroups C andY. Other preferred compositions comprise saccharides from serogroups C,W135 and Y.

The invention provides an immunogenic composition comprising a serogroupA oligosaccharide conjugate and a serogroup C oligosaccharide conjugate,and further comprising (i) an aluminium phosphate or an aluminiumhydroxide adjuvant and (ii) a buffer. Where the composition comprises analuminium phosphate adjuvant, the buffer is preferably a phosphatebuffer; where it comprises an aluminium hydroxide adjuvant, the bufferis preferably a histidine buffer.

Where the vaccine comprises capsular saccharide from serogroup A, it ispreferred that the serogroup A saccharide is combined with the othersaccharide(s) shortly before use, in order to minimise its hydrolysis(cf. Hib saccharides). This can conveniently be achieved by having theserogroup A component in lyophilised form and the other serogroupcomponent(s) in liquid form, with the liquid component being used toreconstitute the lyophilised component when ready for use. The liquidcomponent preferably comprises an aluminium salt adjuvant, whereas thelyophilised serogroup A component may or may not comprise an aluminiumsalt adjuvant.

Thus the invention provides a kit comprising: (a) capsular saccharidefrom N. meningitidis serogroup A, in lyophilised form; and (b) capsularsaccharide(s) from one or more (e.g. 1, 2, 3) of N. meningitidisserogroups C, W135 and Y, in liquid form. The saccharides are preferablyconjugated to carrier protein(s) and/or are oligosaccharides. The kitmay take the form of two vials.

The invention also provides a method for preparing a vaccine compositionof the invention, comprising mixing a lyophilised capsular saccharidefrom N. meningitidis serogroup A with capsular saccharide(s) from one ormore (e.g. 1, 2, 3) of N. meningitidis serogroups C, W135 and Y, whereinsaid one or more saccharides are in liquid form.

The invention also provides a kit comprising: (a) conjugated capsularoligosaccharide from N. meningitidis serogroup A, in lyophilised form;and (b) one or more further antigens in liquid form. The further antigenmay or may not be conjugated capsular oligosaccharide from N.meningitidis serogroup C.

Immunogenic Compositions and Vaccines

Polysaccharides, oligosaccharides and conjugates of the invention areparticularly suited to inclusion in immunogenic compositions andvaccines. A process of the invention may therefore include the step offormulating the polysaccharide, oligosaccharide or conjugate as animmunogenic composition or vaccine. The invention provides a compositionor vaccine obtainable in this way.

Immunogenic compositions and vaccines of the invention will, in additionto the meningococcal saccharides, typically comprise ‘pharmaceuticallyacceptable carriers’, which include any carrier that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolised macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,trehalose [121], lipid aggregates (such as oil droplets or liposomes),and inactive virus particles. Such carriers are well known to those ofordinary skill in the art. The vaccines may also contain diluents, suchas water, saline, glycerol, etc. Additionally, auxiliary substances,such as wetting or emulsifying agents, pH buffering substances, and thelike, may be present. A thorough discussion of pharmaceuticallyacceptable excipients is available in ref. 122.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of saccharide antigen, as well as any other of theabove-mentioned components, as needed. By ‘immunologically effectiveamount’, it is meant that the administration of that amount to anindividual, either in a single dose or as part of a series, is effectivefor treatment or prevention. This amount varies depending upon thehealth and physical condition of the individual to be treated, age, thetaxonomic group of individual to be treated (e.g. non-human primate,primate, etc.), the capacity of the individual's immune system tosynthesise antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials. Dosage treatment may be a single dose schedule or amultiple dose schedule (e.g. including booster doses). The vaccine maybe administered in conjunction with other immunoregulatory agents.

The vaccine may be administered in conjunction with otherimmunoregulatory agents.

The vaccine may include an adjuvant. Preferred adjuvants to enhanceeffectiveness of the composition include, but are not limited to: (1)aluminium salts (alum), such as aluminium hydroxides (includingoxyhydroxides), aluminium phosphates (including hydroxyphosphates),aluminium sulfate, etc [Chapters 8 & 9 in ref 123]; (2) oil-in-wateremulsion formulations (with or without other specific immunostimulatingagents such as muramyl peptides [Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc.] or bacterial cell wall components), such as for example(a) MF59™[Chapter 10 in ref 123; 124, 125], containing 5% Squalene, 0.5%TWEEN®80 (polyoxyethylenesorbitan, monooleate), and 0.5% SPAN®85(sorbitan trioleate)(optionally containing MTP-PE) formulated intosubmicron particles using a microfluidizer, (b) SAF, containing 10%Squalane, 0.4% TWEEN®80 (polyoxyethylenesorbitan, monooleate), 5%PLURONIC™ L121 (block copolymer of propylene oxide and ethylene oxide),and thr-MDP either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion, and (c) RIBI™ adjuvantsystem (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% TWEEN®80 (polyoxyethylenesorbitan, monooleate), and one or morebacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (DETOX™); (3) saponin adjuvants[chapter 22 of ref. 123], such as QS21 or STIMULON™ (CambridgeBioscience, Worcester, Mass.), either in simple form or in the form ofparticles generated therefrom such as ISCOMs (immunostimulatingcomplexes; chapter 23 of ref. 123), which ISCOMS may be devoid ofadditional detergent e.g. ref. 126; (4) Complete Freund's Adjuvant (CFA)and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such asinterleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [127],etc.), interferons (e.g. gamma interferon), macrophage colonystimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6)monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g. refs.128 & 129, optionally in the substantial absence of alum when used withpneumococcal saccharides e.g. ref. 130; (7) combinations of 3dMPL with,for example, QS21 and/or oil-in-water emulsions e.g. refs. 131, 132 &133; (8) oligonucleotides comprising CpG motifs (Roman et al., Nat.Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837;Davis et al., J. Immunol., 1998, 160, 870-876; Chu et al., J. Exp. Med.,1997, 186, 1623-1631; Lipford et al., Eur. J. Immunol., 1997, 27,2340-2344; Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg etal., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93,2879-2883; Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery etal., J. Immunol, 1996, 156, 4570-4575; Halpern et al., Cell Immunol.,1996, 167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79,866-873; Stacey et al., J. Immunol., 1996, 157, 2116-2122; Messina etal., J. Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996,157, 4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al.,J. Immunol., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998,160, 5898-5906; International patent applications WO96/02555,WO98/16247, WO98/18810, WO98/40100, WO98/55495, WO98/37919 andWO98/52581) i.e. containing at least one CG dinucleotide, with5-methylcytosine optionally being used in place of cytosine; (8) apolyoxyethylene ether or a polyoxyethylene ester e.g. ref. 134; (9) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol [135] or a polyoxyethylene alkyl ether or ester surfactant incombination with at least one additional non-ionic surfactant such as anoctoxynol [136]; (10) a saponin and an immunostimulatory oligonucleotide(e.g. a CpG oligonucleotide) [137]; (11) an immunostimulant and aparticle of metal salt e.g. ref. 138; (12) a saponin and an oil-in-wateremulsion e.g. ref. 139; (13) a saponin (e.g. QS21)+3dMPL+IL-12(optionally+a sterol) e.g. ref. 140; (14) E. coli heat-labileenterotoxin (“LT”), or detoxified mutants thereof, such as the K63 orR72 mutants [e.g. Chapter 5 of ref. 141]; (15) cholera toxin (“CT”), ordetoxified mutants thereof [e.g. Chapter 5 of ref. 141]; (16) liposomes[chapters 13 & 14 of ref. 123]; (17) chitosan [e.g. ref. 142]; (18)double-stranded RNA; (19) microparticles (i.e. a particle of ˜100 nm to˜150 μm in diameter, more preferably ˜200 nm to ˜30 μm in diameter, andmost preferably ˜500 nm to ˜10 μm in diameter) formed from materialsthat are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid) suchas poly(lactide-co-glycolide), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone etc.). optionallytreated to have a negatively-charged surface (e.g. with SDS) or apositively-charged surface (e.g. with a cationic detergent, such asCTAB); or (20) other substances that act as immunostimulating agents toenhance the effectiveness of the composition [e.g. chapter 7 of ref.123].

Aluminium salts (especially aluminium phosphates and/or hydroxides) andMF59™ are preferred for use with the saccharide antigens of the presentinvention. Where an aluminium phosphate it used, it is possible toadsorb one or more of the saccharides to the aluminium salt, but it ispreferred not to adsorb the saccharides to the salt, and this isfavoured by including free phosphate ions in solution (e.g. by the useof a phosphate buffer). Where an aluminium hydroxide is used, it ispreferred to adsorb the saccharides to the salt. The use of aluminiumhydroxide as adjuvant is particularly advantageous for saccharide fromserogroup A.

It is possible in compositions of the invention to adsorb some antigensto an aluminium hydroxide but to have other antigens in association withan aluminium phosphate. For tetravalent N. meningitidis serogroupcombinations, for example, the following permutations are available:

Serogroup Aluminium salt (H = a hydroxide; P = a phosphate) A P H P H HH P P P H H H P P P H C P H H P H H P H H P P H P H P P W135 P H H H P HH P H H P P P P H P Y P H H H H P H H P P H P H P P P

For trivalent N. meningitidis serogroup combinations, the followingpermutations are available:

Aluminium salt Serogroup (H = a hydroxide; P = a phosphate) C P H H H PP P H W135 P H H P H P H P Y P H P H H H P P

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals; inparticular, human subjects can be treated. The vaccines are particularlyuseful for vaccinating children and teenagers. They may be delivered bysystemic and/or mucosal routes.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect. Direct delivery of thecompositions will generally be parenteral (e.g. by injection, eithersubcutaneously, intraperitoneally, intravenously or intramuscularly ordelivered to the interstitial space of a tissue). The compositions canalso be administered into a lesion. Other modes of administrationinclude oral and pulmonary administration, suppositories, andtransdermal or transcutaneous applications (e.g. see ref. 143), needles,and hyposprays. Dosage treatment may be a single dose schedule or amultiple dose schedule (e.g. including booster doses).

Vaccines of the invention are preferably sterile. They are preferablypyrogen-free. They are preferably buffered e.g. at between pH 6 and pH8, generally around pH 7. Where a vaccine comprises an aluminiumhydroxide salt, it is preferred to use a histidine buffer [144].

Vaccines of the invention may comprise detergent (e.g. a Tween, such asTween 80) at low levels (e.g. <0.01%). Vaccines of the invention maycomprise a sugar alcohol (e.g. mannitol) or trehalose e.g. at around 15mg/ml, particularly if they are to be lyophilised.

Optimum doses of individual antigens can be assessed empirically. Ingeneral, however, saccharide antigens of the invention will beadministered at a dose of between 0.1 and 100 μg of each saccharide perdose, with a typical dosage volume of 0.5 ml. The dose is typicallybetween 5 and 20 μg per saccharide per dose. These values are measuredas saccharide.

Vaccines according to the invention may either be prophylactic (i.e. toprevent infection) or therapeutic (i.e. to treat disease afterinfection), but will typically be prophylactic.

The invention provides a method of raising an immune response in apatient, comprising administering to a patient a vaccine according tothe invention. The immune response is preferably protective againstmeningococcal disease, and may comprise a humoral immune response and/ora cellular immune response. The patient is preferably a child.

The method may raise a booster response, in a patient that has alreadybeen primed against N. meningitidis.

The invention also provides the use of a polysaccharide, oligosaccharideor conjugate of the invention in the manufacture of a medicament forraising an immune response in an animal. The medicament is preferably animmunogenic composition (e.g. a vaccine). The medicament is preferablyfor the prevention and/or treatment of a disease caused by a Neisseria(e.g. meningitis, septicaemia, gonorrhoea etc.), by H. influenzae (e.g.otitis media, bronchitis, pneumonia, cellulitis, pericarditis,meningitis etc.) or by pneumococcus (e.g. meningitis, sepsis, pneumoniaetc). The prevention and/or treatment of bacterial meningitis is thuspreferred.

Vaccines can be tested in standard animal models (e.g. see ref. 145).

The invention also provides a process for solubilising a precipitatedbacterial capsular polysaccharide, wherein ethanol is used as a solvent.

DEFINITIONS

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of varying ethanol:water ratios onpolysaccharide solubilisation.

FIGS. 2 to 4 show IgG titres obtained in mice against oligosaccharideantigens:

FIG. 2 shows results using serogroup A oligosaccharide;

FIG. 3 shows results for serogroup Y; and

FIG. 4 shows results for serogroup W135.

FIG. 5 shows post-II IgG titres obtained in mice with a mixture ofoligosaccharide conjugates for serogroups A and C: FIG. 5 a shows theanti-serogroup A responses; and FIG. 5 b shows anti-serogroup Cresponses.

FIGS. 6 to 8 show IgG titres obtained in mice with a mixture ofoligosaccharide conjugates for serogroups C, W135 and Y:

FIG. 6 shows the anti-serogroup W135 responses;

FIG. 7 shows anti-serogroup Y responses; and

FIG. 8 shows anti-serogroup C responses.

FIGS. 9 to 11 show post-II IgG titres obtained in mice with a mixture ofoligosaccharide conjugates for serogroups A, C, W135 and Y:

FIG. 9 shows the anti-serogroup W135 responses;

FIG. 10 shows anti-serogroup Y responses; and

FIG. 11 shows anti-serogroup A responses.

FIG. 12 is a calibration curve obtained using test MenA polysaccharidesamples at different hydrolysis times. The curve shows the linearrelationship between the reciprocal of the degree of polymerisation andoptical rotatory power.

FIG. 13 is a calibration curve obtained using test MenY polysaccharidesamples at different hydrolysis times. The curve shows the linearrelationship between the log of the degree of polymerisation and KD(distribution coefficient).

FIGS. 14 to 16 show post-II IgG titres, split by IgG subclass, obtainedin mice after immunisation with oligosaccharide conjugates forserogroups: (14) A; (15) C; (16) W135 and (17) Y.

FIG. 17 shows post-II IgG titres, split by IgG subclass, obtained inmice after immunisation with a tetravalent mixture of oligosaccharideconjugates. FIG. 17(A) shows the results of IgG subclass analysis forMenA. FIG. 17(B) shows the results of IgG subclass analysis for MenC.FIG. 17(C) shows the results of IgG subclass analysis for MenW135. FIG.17(D) shows the results of IgG subclass analysis for MenY.

FIG. 18 illustrates the preparation of an oligosaccharide conjugate.

FIG. 19 shows (A) anti-MenA and (B) anti-MenC GMT (±95% confidenceintervals) obtained in a guinea pig model. Values above bars are serumbactericidal assay (SBA) titres i.e. the reciprocal of the sera dilutionyielding the 50% of killing.

MODES FOR CARRYING OUT THE INVENTION A. Production and Purification ofMeningococcal Polysaccharides

Meningococci of serogroups A, W135 and Y were grown in 500 ml flaskscontaining 150 ml of Franz A as medium, for 12 hours at 35±1° C.Agitation was set at 150 rpm using a 35 mm throw Shaker. 85 ml of theculture was then inoculated in 20 L fermentor containing Watson asmedium.

After 18.5 hours (W135 and Y) or 16.5 hours (A), when OD=10 was reached,the fermentation was interrupted by adding 300 ml of formalin and then,after 2 hours of incubation, and the fermentor was cooled to 10° C. Thesupernatant was collected by centrifugation followed by filtration (0.22μm), and ultrafiltration with a 30 kDa membrane.

The crude concentrated polysaccharide was then precipitated by additionof CTAB as a 100 mg/ml water solution. The volumes added are shown inthe following table. After 12 hours at room temperature, the CTABcomplexes were recovered by centrifugation. The CTAB complex wasextracted by adding a 95% ethanol solution at room temperature for 16-20hrs under vigorous stirring. The volume of ethanol added is shown in thefollowing table:

CTAB volume Volume of 95% ethanol Serogroup (ml) (liters per kg wetpaste) A 475 3.5 to 6   W135 200 4 to 6 Y 650 3.4

The resulting suspensions were filtered through a CUNO 10 SP depthfilter. The filtrate was recirculated through a CUNO ZETACARBON™cartridge until OD_(275nm)<0.2. The Z carbon filtrate was then collectedand filtered through a 0.22 μm filter. The polysaccharide was eventuallyprecipitated from the ethanol phase by addition of a CaCl₂ 2M watersolution (10-12 ml/l of EtOH final solution). The purifiedpolysaccharide was then collected by centrifugation, washed with 95%ethanol and dried under vacuum.

In other experiments, the final concentration of ethanol used forextraction was varied (FIG. 1). For serogroup A polysaccharide, a rangeof between 80 and 95% ethanol was most effective, with extractionefficiency decreasing at lower percentages. For serogroup W135, goodextraction was achieved with between 75% and 90% ethanol, with 95% beingless effective. For serogroup Y, the best results were achieved withbetween 75% and 85% ethanol, with higher percentages (e.g. 90%, 95%)being less effective. In general, it was noted that ethanol percentagesbelow those reported here tended to increase the co-extraction ofcontaminants such as proteins. Ethanol percentages given in thisparagraph are expressed as a final concentration (ethanol as percentageof total volume of ethanol+water) and are based on a water content inthe CTAB-polysaccharide pastes recovered by centrifugation of about 50%(i.e. 500 g H₂O per kg wet paste). This value was determined empiricallyin small scale-up experiments.

B. Conjugation of Serogroup A Polysaccharides

a) Hydrolysis

The serogroup A meningococcal polysaccharide was hydrolysed in 50 mMsodium acetate buffer, pH 4.7 for about 3 hrs at 73° C. The hydrolysiswas controlled in order to obtain oligosaccharides with an averagedegree of polymerisation (DP) of approximately 10, as determined by the(w/w) ratio between the total organic phosphorus and the monoesterphosphate.

The DP ratio of (total organic phosphorus) to (phosphorus monoester) isinversely proportional to optical rotatory power (α), as shown in FIG.12. This relationship can be used to monitor the extent of hydrolysismore conveniently than direct phosphorus measurements.

b) Sizing

This step removes short-length oligosaccharides generated during thehydrolysis process. The hydrolysate obtained above was ultrafilteredthrough a 30 kDa cut-off membrane (12 diafiltration volumes of 5 mMacetate buffer, pH 6.5). The retentate, containing the high Mw species,was discarded; the permeate was loaded onto a onto a Q-Sepharose FastFlow column equilibrated in acetate buffer 5 mM, pH 6.5. The column wasthen washed with 5 column volumes (CV) of equilibrating buffer, thenwith 10 CV of 5 mM acetate buffer/125 mM NaCl pH 6.5 in order to removeoligosaccharides with DP≦6. The sized oligosaccharide was then elutedwith 5 CV of 5 mM acetate buffer/0.5 M NaCl pH 6.5.

The eluted oligosaccharide population has an average DP of about 15.

c) Introduction of a Primary Amino Group at the Reducing Terminus

Ammonium salt (acetate or chloride) was added to the sizedoligosaccharide solution for a final concentration ranging from 49-300g/L, then sodium-cyano-borohydride was added to a final concentrationranging from 12-73 g/L. After adjusting the pH to between 6-7.3, themixture was incubated at 37° C. for 5 days.

The amino-oligosaccharides were then purified by tangential flowultrafiltration with a 1 kDa or 3 kDa cut-off membrane using 13diafiltration volumes of 0.5 M NaCl followed by 7 diafiltration volumesof 20 mM NaCl. The purified amino-oligosaccharide solution was analysedfor phosphorus content (one chemical activity of the antigen) by theprocedure of ref. 146 and the amount of introduced amino groups by theprocedure of ref. 147.

The purified oligosaccharides were then dried with rotary evaporator toremove water.

d) Derivatisation to Active Ester

The dried amino-oligosaccharides were solubilised in distilled water ata 40 mM amino group concentration, then 9 volumes of DMSO were addedfollowed by triethyl-amine at a final concentration of 200 mM. To theresulting solution, adipic acid N-hydroxysuccinimido diester was addedfor a final concentration of 480 mM.

The reaction was maintained under stirring at room temperature for 2hours, then the activated oligosaccharide was precipitated with acetone(80% v/v final concentration). The precipitate was collected bycentrifugation and washed several times with acetone to remove unreactedadipic acid N-hydroxysuccinimido diester and by-products. Finally theactivated oligosaccharide was dried under vacuum.

The amount of active ester groups introduced into the oligosaccharidestructure was determined by a colorimetric method as described in ref.148.

e) Conjugation to CRM₁₉₇

The dried activated oligosaccharide was added to a 45 mg/ml solution ofCRM₁₉₇ in 0.01M phosphate buffer pH 7.2 for an active ester/protein(mole/mole) ratio of 12:1. The reaction was maintained under stirring atroom temperature overnight. After this period, the conjugate waspurified by hydrophobic chromatography or tangential flowultrafiltration. The purified MenA-CRM₁₉₇ conjugate was sterile filteredand stored at −20° C. or −60° C. until vaccine formulation.

The conjugate was analysed for: protein content (microBCA ProteinAssay), MenA saccharide content (colorimetric analysis of phosphorus),free saccharide content, HPLC profile (on TSKgel G4000SW 7.5 mm IDx30cm), and SDS-PAGE. Characteristics of typical preparations are shown inthe following table:

Saccharide Lot Code (mg/ml) protein (mg/ml) Glycosylation KD 210201/A0.257 0.864 0.3 0.489 210201/BS 0.308 1.354 0.23 0.503 210201/BL 0.281.482 0.19 0.501 35I230595 0.138 0.3 0.46 010900 0.092 0.337 0.27 DP290.105 0.245 0.43 A1 (UNSIZED) 0.08 0.291 0.27 A2 (SIZED) 0.446 2.4210.18

C. Conjugation of Serogroup W135 Polysaccharides

a) Hydrolysis

The group W meningococcal polysaccharide was hydrolysed in acetic 50 mMsodium acetate buffer, pH 4.7 for about 3 hours at 80° C. This resultedin oligosaccharides with an average DP of about 15 to 20 as determinedby ratio between sialic acid (SA) and reduced terminal SA.

The DP ratio of (total SA) to (reduced terminal SA) is related to the KDof the as determined by HPLC-SEC, as shown in FIG. 13. This relationshipcan be used to monitor the extent of hydrolysis more conveniently thandirect SA measurements.

b) Sizing

The hydrolysate was ultrafiltered through a 30 kDa cut-off membrane (12to 20 diafiltration volumes of 5 mM acetate buffer/15-30 mM NaCl pH6.5). The retentate, containing the high MW species, was discarded whilethe permeate was loaded onto a Q-Sepharose Fast Flow column equilibratedin 5 mM acetate buffer/15 mM NaCl pH 6.5. The column was then washedwith 10 CV equilibrating buffer, in order to remove oligosaccharideswith DP≦3-4 and eluted with 3 CV 5 mM acetate buffer/500 mM NaCl pH 6.5.

c) Introduction of a Primary Amino Group at the Reducing Terminus

Ammonium chloride or ammonium acetate was added to the sizedoligosaccharide solution to a final concentration of 300 g/L, thensodium-cyano-borohydride was added to 49 g/L or 73 g/L finalconcentration. The mixture was incubated at 50° C. for 3 days.

The amino-oligosaccharides were then purified by tangential flowultrafiltration as described for serogroup A. The purified material wasanalysed for its content of sialic acid (colorimetric method accordingto ref. 149 and/or galactose (HPLC) (chemical activities of the MenW135antigen). The purified oligosaccharides were then dried with rotaryevaporator to remove water.

d) Derivatisation to Active Ester

The dried amino-oligosaccharides were derivatised as described above forserogroup A.

e) Conjugation to CRM₁₉₇

Conjugation was performed as described above for serogroup A but, topurify the conjugate, diafiltration with a 30 kDa membrane was used (50diafiltration volumes of 10 mM phosphate buffer, pH 7.2). The purifiedconjugate was sterile filtered and stored at −20° C. or −60° C. untilvaccine formulation.

The conjugate was analysed for the same parameters as described abovefor serogroup A. MenW saccharide content was assayed by calorimetricsialic acid determination:

protein Lot code saccharide (mg/ml) (mg/ml) Glycosylation KD lot 1 5.733.52 1.63 0.296 lot 2/4, 5 3.51 2.88 1.22 0.308 lot 3S 2.49 2.25 1.110.380 lot 3Sd 2.03 2.24 0.91 0.394 lot 3L 2.32 2.3 1.01 0.391 lot 3Ld1.94 2.29 0.85 0.383 Lot 3S/pr. Glic6 0.363 0.82 0.44 0.498 Lot 3S/pr.Glic9 0.424 0.739 0.57 0.447 Lot 3S/pr. Glic12 0.479 0.714 0.671 0.414

D. Conjugation of Serogroup Y Polysaccharides

a) Hydrolysis

The group Y meningococcal polysaccharide was hydrolysed as describedabove for serogroup W135. This gave oligosaccharides with an average DPof about 15 to 20 as determined by ratio between SA and reduced terminalSA (conveniently measured indirectly as described under C(a) above).

b) Sizing, c) Introduction of Amino Group, d) Derivatisation to ActiveEster and e) Conjugation

These steps were performed as described above for serogroup W135. Thepurified conjugate was sterile filtered and stored at −20° C. or −60° C.until vaccine formulation.

The conjugate was analysed in the same way as described above forserogroup W135:

Lot Code saccharide (mg/ml) protein (mg/ml) Glycosylation KD lot 1A 1.160.92 1.26 0.303 lot 1B 4.57 3.55 1.29 0.339 Lot 2/4, 5 2.32 6.1 0.380.467 lot 2/6 1.75 5.73 0.3 0.498

E. Immunogenicity of Individual Conjugates

The frozen bulk conjugates were thawed. Each was diluted, understirring, to a final concentration of 20 μg saccharide/ml, 5 mMphosphate, 9 mg/ml NaCl, aluminium phosphate (to give anAl³⁺concentration of 0.6 mg/ml), pH 7.2. The mixtures were then kept,without stirring, at 2-8° C. overnight and further diluted with salineto 4 μg saccharide/ml for mouse immunisation.

A second set of vaccines was prepared for each serogroup in the sameway, but the addition of aluminium phosphate was replaced with samevolume of water.

Ten Balb/c mice for each immunisation group were injected s.c. twicewith 0.5 ml vaccine at weeks 0 and 4. Bleedings were performed beforeimmunisation, the day before the second dose and 2 weeks after thesecond dose. Immunisations were performed with (a) the conjugate vaccinewith or without alum, (b) saline control and (c) unconjugatedpolysaccharide control.

Specific anti-polysaccharide IgG antibodies were determined in the seraof immunised animals essentially as described in ref. 150. Eachindividual mouse serum was analysed in duplicate by a titration curveand GMT was calculated for each immunisation group. Titres werecalculated in Mouse Elisa Units (MEU) using ‘Titerun’ software (FDA).Anti-polysaccharide titre specificity was determined by competitiveELISA with the relevant polysaccharide as competitor.

As shown in FIG. 2, the MenA conjugate induced high antibody titres inanimals. As expected, the unconjugated polysaccharide was notimmunogenic. The conjugate formulation with an aluminium phosphate asadjuvant induced a higher level of antibodies compared to the titreobtained by the conjugate alone. Similar results were seen for MenY(FIG. 3) and MenW135 (FIG. 4).

The IgG subclass of the post-II immune responses was measured forvarious groups. Specific subclasses were determined using the same ELISAmethod as used for the determination of the total IgG titer in section Eabove, but using alkaline phosphatase-anti mouse-IgG1, -IgG2a, -IgG2b or-IgG3 (Zymed) as the secondary antibody. Titres were expressed asOD_(405nm) obtained after 30 minutes of substrate development usingserum diluted 1:3200, and are shown in FIGS. 14 (MenA), 15 (MenW135) and16 (MenY). Responses are primarily in subclass IgG1, which is thesubclass predominantly induced in mice by T-dependent antigens. Becausepolysaccharides are inherently T-independent antigens which are not ableto induce immunological memory, these data show that conjugation has hadthe desired effect.

Post-II sera were also tested for bactericidal activity using an invitro assay to measure complement-mediated lysis of bacteria. Post-IIsera were inactivated for 30 minutes at 56° C. before the use in theassay, and 25% baby rabbit complement was used as source of complement.Bactericidal titre was expressed as the reciprocal serum dilutionyielding 50% killing of bacteria against the following strains: MenAG8238, A1, F6124; MenW135 5554(OAc+) and 242317(OAc−); MenY 242975(OAc−)and 240539(OAc+).

Results for MenA included:

Poly/oligo Approx. Aluminium Carrier saccharide αDP adjuvant GMTBactericidal activity CRM₁₉₇ O 15 — 461 F8238: 2048-4096; F6124:2048-4096 CRM₁₉₇ O 15 phosphate 920 F8238: 4096; F6124: 4096 — P —phosphate 3 F8238: 8; F6124: 128 CRM₁₉₇ O 15 — 290 F8238: 512-1024 — P —— 2 F8238: <4 CRM₁₉₇ O 15 — 155 F8238: 512-1024 CRM₁₉₇ O 15 — 393 F8238:1024 CRM₁₉₇ O 15 — 396 — CRM₁₉₇ O 15 phosphate 1396 F8238: 4096 CRM₁₉₇ O15 phosphate 1461 F8238: 2048-4096 CRM₁₉₇ O 15 phosphate 1654 F8238:2048 CRM₁₉₇ O 29 phosphate 1053 F8238: 2048 CRM₁₉₇ unsized O 10phosphate 1449 F8238: 2048 CRM₁₉₇ O 15 phosphate 626 F8238: 2048-4096CRM₁₉₇ O 15 — 742 — CRM₁₉₇ O 15 — 2207 — CRM₁₉₇ O 29 — 1363 — CRM₁₉₇unsized O 10 — 615 — CRM₁₉₇ O 15 phosphate 1515 — CRM₁₉₇ O 15 phosphate876 — CRM₁₉₇ O 15 phosphate 1232 — CRM₁₉₇ O 15 phosphate 852 — CRM₁₉₇ O15 phosphate 863 F8238: 2048; A1: 2048; F6124: >2048 CRM₁₉₇ O 27phosphate 1733 F8238: 4096-8192; F6124: 4096-8192 CRM₁₉₇ O 15 phosphate172 F8238: 1024; A1: 1024-2048; F6124: 2048 CRM₁₉₇ O 15 hydroxide 619F8238: 1024; A1: 2048; F6124: 2048

Results for MenW135 included:

Poly/oligo Aluminium Carrier saccharide OAc adjuvant GMT Bactericidalactivity CRM₁₉₇ O + — 14 5554: 256-512 CRM₁₉₇ O + phosphate 23 5554:256-512 — P — — 5554: 4 CRM₁₉₇ O + — 45 5554: 1024 CRM₁₉₇ O + — 1015554: 64-128 CRM₁₉₇ O + — 80 5554: 256-512 CRM₁₉₇ O + phosphate 2215554: 1024-2048; 242317: 1024-2048 CRM₁₉₇ O − — 52 5554: 512-1024 CRM₁₉₇O − phosphate 329 5554: 1024-2048; 242317: 1024-2048 CRM₁₉₇ O + — 415554: 256-512 CRM₁₉₇ O + phosphate 24 5554: 1024; 242317: 128-256 CRM₁₉₇O − — 116 5554: 256-512 CRM₁₉₇ O − phosphate 185 5554: 1024; 242317:512-1024 CRM₁₉₇ O + phosphate 565 5554: 2048 CRM₁₉₇ O + phosphate 3285554: 512-1024 CRM₁₉₇ O + phosphate 490 5554: 1024-2048 CRM₁₉₇ O +hydroxide 189 5554: 512-1024; 242317: 512-1024 CRM₁₉₇ O + phosphate 805554: 512-1024; 242317: 512-1024 CRM₁₉₇ O + hydroxide 277 5554:512-1024; 242317: 1024-2048

Results for MenY included:

Poly/oligo Aluminium Carrier saccharide αDP adjuvant GMT Bactericidalactivity CRM₁₉₇ O >15 — 751 242975: 8192 CRM₁₉₇ O >15 phosphate 1190242975: 8192-16384; 240539: 8192-16384 CRM₁₉₇ O >15 — 284 242975:2048-4096 CRM₁₉₇ O >15 phosphate 775 242975: 2048-4096 — P — — — 242975:256 CRM₁₉₇ O >15 — 1618 242975: 4096-8192 CRM₁₉₇ O >15 — 2123 242975:2048 CRM₁₉₇ O <10 — 253 242975: 512-1024 CRM₁₉₇ O <10 — 1060 242975:256-512 CRM₁₉₇ O >15 hydroxide 1167 242975: 8192; 240539: 8192-16384CRM₁₉₇ O >15 phosphate 665 242975: 8192; 240539: 8192-16384 CRM₁₉₇ O >15phosphate 328 242975: 4096; 240539: 2048-4096 CRM₁₉₇ O >15 hydroxide 452242975: 2048; 240539: 1024-2048

F. Immunogenicity of MenA Conjugate in Combination with MenC Conjugate

CRM-MenC concentrated bulk (from Chiron Vaccines, Italy) was mixed withCRM-MenA concentrated bulk (obtained as described above) were dilutedand mixed by stirring. Three different preparations were made. Eachcontained 20 μg saccharide/ml for MenA, but different amounts of MenCconjugate were included: (i) 20 μg saccharide/ml (ii) 10 μgsaccharide/ml; (iii) 5 μg saccharide/ml. Ratios of MenA:MenC (w/w) werethus: (i) 1:1; (ii) 2:1; (iii) 4:1.

Each preparation also contained 5 mM sodium phosphate, 9 mg/ml NaCl,aluminium phosphate (to give an Al³⁺ concentration of 0.6 mg/ml), pH7.2. Each mixture was then kept, without stirring, at 2-8° C. overnightand further diluted 1:5 with saline before mice immunisation.

A second set of vaccines was prepared in the same way, but the additionof aluminium phosphate was replaced with same volume of water.

For each of the six vaccines, ten Balb/c mice were immunised asdescribed above. Control groups received saline or MenA conjugate alone.

Anti-polysaccharide antibodies for MenA and MenC were determined asdescribed above.

The results obtained with the mixture of MenA+MenC conjugates clearlyindicate that the ratio (w/w) between A and C components plays a crucialrole for MenA immunogenicity.

The specific anti-MenApS titre obtained with the MenA conjugate controlwas higher (with or without alum adjuvant) than for the MenA+MenCcombination at the same dosage (FIG. 5 a). When a lower amount of MenCconjugate is used in the combination, a better anti-MenApS titre isinduced by the MenA conjugate component. At the same time, the anti-MenCtitre remains acceptable (FIG. 5 b).

Experiments were also performed using a guinea pig model. Threedifferent preparations were made, using the same aluminium phosphateadjuvant as before (amorphous hydroxyphosphate, PO₄/Al molar ratiobetween 0.84 and 0.92, 0.6 mg Al³⁺/ml):

Men A:Men C Preparation Men A* Men C* ratio A 20 μg/ml 20 μg/ml 1:1 B 40μg/ml 20 μg/ml 2:1 C 20 μg/ml 10 μg/ml 1:½ *Expressed as saccharide

These preparations were diluted 1:2 with saline and used to immuniseguinea pigs. Five guinea pigs (Hartelley strain, female, 450-500 grams)for each immunisation group were injected s.c. twice with 0.5 ml vaccineat days 0 and 28. Bleedings were performed before the first immunisationand then at day 42. Sera were stored at −70° C. prior to analysis byELISA and serum bactericidal assay (against MenA strain MK 83/94 or.MenC strain C11). Results are shown in FIG. 19.

G. Combination Vaccine for Serogroups C, W135 and Y

Conjugates of polysaccharides from serogroups C, W135 and Y were mixedas described above to give a final concentration of 20 μg saccharide/mlfor each conjugate. The vaccine contained a final concentration of 5 mMsodium phosphate and 9 mg/ml NaCl, pH 7.2. After overnight storage, themixture was diluted to contain 4 μg saccharide/ml for each conjugate forimmunisation.

Immunisations and analysis took place as before.

The results show that the immunogenicity of MenW135 conjugate isenhanced when administered in combination with MenC and MenY conjugates,when compared to that obtained with the MenW135 conjugate alone (FIG.6). MenY immunogenicity was comparable in the combination to thatobtained with the individual conjugate (FIG. 7) and was also comparableto the immunogenicity of the MenC conjugate (FIG. 8).

H. Combination Vaccine for Serogroups A, C, W135 and Y

Conjugates of polysaccharides from serogroups A, C, W135 and Y weremixed as described above to give a final concentration of 20 μgsaccharide/ml for the serogroup A, W135 and Y conjugates and 5 μgsaccharide/ml for the serogroup C conjugate. The vaccine contained afinal concentration of 5 mM sodium phosphate, 9 mg/ml NaCl, aluminiumphosphate (to give an Al³⁺ concentration of 0.6 mg/ml), pH 7.2. Themixture was then kept, without stirring, at 2-8° C. overnight andfurther diluted with saline to give 4 μg saccharide/ml for the A, W135and Y conjugates and 1 μg saccharide/ml for the C conjugate. Thisdiluted mixture was used for immunisation.

Immunisations and analysis took place as before, with controls includingthe individual conjugates except for serogroup C.

FIG. 9 shows that, as before, the immunogenicity of the MenW135conjugate was enhanced when administered in combination with the MenA,MenC and MenY conjugates. FIG. 10 shows that the immunogenicity of theMenY conjugate is not significantly different when delivered incombination with the MenA, MenC and MenW135 conjugates. FIG. 11 showsthat the immunogenicity of the MenA conjugate decreases markedly in thecombination, even with the MenC conjugate administered at a lower dosage(¼). This antigenic competition is not seen in the non-conjugatedtetravalent (ACWY) polysaccharide vaccine [5].

I. Lyophilised Serogroup A Antigen

The capsular polysaccharide of serogroup A N. meningitidis isparticularly susceptible to hydrolysis. Conjugates of MenA capsularoligosaccharide were therefore prepared in lyophilised form, ready forre-constitution at the time of administration. The lyophilised form wasprepared to have components which give the following composition afterreconstitution into a unit dose:

Component Concentration CRM-Men A 20 μg saccharide/ml Potassiumphosphate buffer  5 mM Mannitol 15 mg/ml

This composition has no adjuvant. Two adjuvants were prepared for itsreconstitution:

Component Concentration Concentration Aluminium hydroxide 0.68 mgAl³⁺/ml — Aluminium phosphate* — 0.6 mg Al³⁺/ml Sodium phosphate buffer— 10 mM Histidine buffer 10 mM — Sodium chloride 9 mg/ml 9 mg/ml Tween80 0.005% 0.005% PH 7.2 ± 0.05 7.2 ± 0.05 *amorphous hydroxyphosphate,PO₄/Al molar ratio between 0.84 and 0.92

When reconstituted with water for injection, stability of the saccharidecomponent was as follows:

Stored at 2-8° C. Stored at 36-38° C. Total Free Total Free Timesaccharide saccharide Free saccharide saccharide Free (days) (μg/ml)(μg/ml) saccharide % (μg/ml) (μg/ml) saccharide % 0 17.72 1.04 5.9 17.721.04 5.9 15 17.01 0.88 5.2 16.52 2.26 13.7 30 17.82 0.89 5.0 17.29 2.6415.3

Over the same 4 week time scale, pH was stable at 7.2 both at 2-8° C.and at 36-38° C., protein content was stable at around 24.5 μg/ml, andmoisture content was below 2.5%.

When reconstituted with the aluminium phosphate adjuvant solution at andstored at 2-8° C., stability was as follows:

Free Free Time Total saccharide saccharide saccharide (hours) (μg/ml)(μg/ml) % 0 16.62 1.09 6.6 24 16.51 0.98 5.9 48 16.83 0.99 5.9

J. Combination Vaccine for Serogroups A, C, W135 and Y LyophilisedSerogroup A Conjugate

A trivalent mixture of the MenC, W135 and Y components either adsorbedonto an aluminium hydroxide adjuvant (2 mg/ml) or mixed with analuminium phosphate adjuvant (amorphous hydroxyphosphate, PO₄/Al molarratio between 0.84 and 0.92, 0.6 mg/ml Al³⁺, in presence of 10 mMphosphate buffer) was prepared. The compositions of the two trivalentmixtures were as follows:

Component Concentration Concentration Aluminium hydroxide 0.68 mgAl³⁺/ml — Aluminium phosphate* — 0.6 mg Al³⁺/ml CRM-MenC 20 μgsaccharide/ml 20 μg saccharide/ml CRM-MenY 20 μg saccharide/ml 20 μgsaccharide/ml CRM-MenW135 20 μg saccharide/ml 20 μg saccharide/ml Sodiumphosphate buffer — 10 mM Histidine buffer 10 mM — Sodium chloride 9mg/ml 9 mg/ml Tween 80 0.005% 0.005% *amorphous hydroxyphosphate, PO₄/Almolar ratio between 0.84 and 0.92

For the hydroxide mixture, stability of the saccharide components wereas follows:

Stored at 2-8° C. Stored at 36-38° C. Time Free saccharide Free Freesaccharide Free (days) (μg/ml) saccharide % (μg/ml) saccharide % MenCbulk  0 <1.2 <6 <1.2 <6 15 <1.2 <6 <1.2 <6 30 <1.2 <6 <1.2 <6 MenC vials 0 <1.2 <6 <1.2 <6 15 <1.2 <6 <1.2 <6 30 <1.2 <6 1.3 6.6 MenW135 bulk  02.5 12.5 2.5 12.5 15 2.3 11.4 3.4 16.8 30 2.3 11.5 3.5 17.3 MenW135vials  0 2.1 10.6 2.1 10.6 15 2.3 11.7 2.7 13.3 30 20. 10.2 3.3 16.3MenY bulk  0 1.7 8.3 1.7 8.3 15 <1.3 <6.3 2.0 10.2 30 1.3 6.3 2.4 12.2MenY vials  0 1.4 7.1 1.4 7.1 15 1.5 7.6 2.1 10.7 30 1.3 6.3 2.9 14.3

Over the same 4 week time scale, pH was stable at 7.15±0.05 both at 2-8°C. and at 36-38° C.

For the phosphate mixture, stability of the saccharide components wereas follows:

Stored at 2-8° C. Stored at 36-38° C. Total Free Total Free Timesaccharide saccharide Free saccharide saccharide Free (days) (μg/ml)(μg/ml) saccharide % (μg/ml) (μg/ml) saccharide % MenC bulk  0 22.8 <1.0<5 22.8 <1.0 <5 15 17.2 <1.0 <5 18.6 <1.0 <5 30 18.9 <1.0 <5 20.5 <1.0<5 MenC vials  0 20.5 <1.0 <5 20.5 <1.0 <5 15 18.3 <1.0 <5 23.4 <1.0 <530 18.0 <1.0 <5 20.5 <1.0 <5 MenW135 bulk  0 20.7 2.0 10.4 20.7 2.0 10.415 21.9 2.3 11.6 21.2 2.1 10.3 30 19.6 2.1 10.6 21.0 2.4 11.8 MenW135vials  0 23.4 1.7 8.4 23.4 1.7 8.4 15 21.2 1.9 9.5 20.1 2.2 11.1 30 20.12.2 11.2 21.3 3.2 16.1 MenY bulk  0 19.1 <1.1 <5.3 19.1 <1.1 <5.3 1520.1 1.4 6.8 18.7 1.3 6.4 30 18.6 1.4 7.6 19.2 1.7 8.3 MenY vials  021.4 <1.1 <5.3 21.4 <1.1 <5.3 15 19.6 1.4 6.8 19.0 1.5 7.4 30 17.7 1.26.2 18.4 1.9 9.4

Over the same 4 week time scale, pH was stable at 7.05±0.05 both at 2-8°C. and at 36-38° C.

The trivalent liquid compositions were diluted and 0.5 ml used toreconstitute the lyophilised MenA conjugate. The resulting tetravalentmixture was administered to ten Balb/c mice (female 6-8 weeks old) pergroup by subcutaneous injection at day 0 and 28. The mixture contained 2μg of each saccharide conjugate per dose, which represents ⅕ of thesingle human dose (SHD). Controls were saline or unconjugated homologouspolysaccharides. Bleedings were performed before immunization and thenat day 42, with sera stored at −70° C. IgG was determined as describedabove.

All the conjugates used were safe and immunogenic in the animals. GMTpost-II ELISA titres (with 95% confidence intervals) were as follows:

Vaccine Adjuvant A Y W135 C MenA (lyophilised Aluminium 172 — — — andphosphate  (69-439) resuspended) Aluminium 619 — — — hydroxide (419-906)MenY Aluminium — 328 — — phosphate (147-731) Aluminium — 452 — —hydroxide (344-593) MenW Aluminium — —  80 — phosphate (28-225)Aluminium — — 277 — hydroxide (185-411)  MenC Aluminium — — — 317phosphate (152-659) Aluminium — — — 723 hydroxide (615-851) MenA(lyophilized) + Aluminium  32 397  99 114 MenC, W135, Y phosphate(15-68) (252-627) (35-288)  (53-246) Aluminium 206 141 139 163 hydroxide(112-372)  (97-205) (76-251) (122-218)

FIG. 17 shows the results of IgG subclass analysis for: (17A) MenA;(17B) MenC; (17C) MenW135; and (17D) MenY. IgG1 is clearly the mostprominent subclass.

Serum bactericidal titres were as follows:

Anti- Anti- Anti-MenA Anti-MenY MenW135 MenC Vaccine Adjuvant F8238 A1F6124 242975 240539 5554 242317 C11 MenA Aluminium  512-1024 1024-20482048 — — — — — (lyophilised) phosphate Aluminium 1024-2048 1024-20482048 — — — — — hydroxide MenY Aluminium — — — 4096 2048-4096 — — —phosphate Aluminium — — — 2048 1024-2048 — — — hydroxide MenW Aluminium— — — — —  512  512-1024 — phosphate Aluminium — — — — — 1024 1024-2048— hydroxide MenC Aluminium — — — — — — — 2048-4096 phosphate Aluminium —— — — — — — 4096 hydroxide MenA Aluminium 128-256 1024 1024-2048 2048 —256-512 1024  512 (lyophilized) + phosphate MenC, W135, Y Aluminium 5121024-2048 1024-2048 2048-4096 — 256-512 1024  512-1024 hydroxide

K. Combination Vaccine for Serogroups A, C, W135 and Y Different Dosages

Mice were immunised as described above, but the vaccine compositionscontained different ratios of the various oligosaccharide conjugates.Doses were variously 0.5, 1, 2 or 4 μg/dose. Lyophilised MenAoligo-conjugate was used in all experiments.

ELISA titres were as follows:

Antigen quantity (μg/dose) Aluminium GMT ELISA (95% confidence interval)A C W135 Y adjuvant A C W135 Y 4 2 2 2 Phosphate 177 367 239  239(107-291) (263-510) (135-424)  (184-311) 4 2 2 2 Hydroxide 390 494 338 158 (313-486) (345-706) (266-430)   (96-260) 2 2 2 2 Phosphate 132 582143  247  (59-296) (268-1155) (75-272) (152-400) 2 2 2 2 Hydroxide 337569 171  100 (239-476) (462-679) (117-251)  (59-169) 4 2 1 1 Phosphate137 192 18 315  (47-397)  (88-421) (4-75) (174-571) 4 2 1 0.5 Phosphate152 207 51 220  (85-271) (100-428) (21-125) (125-388) 4 2 1 2 Phosphate113 230 23 267  (49-263)  (98-540) (6-91)  (81-877) 4 2 0.5 1 Phosphate267 504 46 583 (109-656) (300-847) (15-134)  (330-1030) 4 2 2 1Phosphate  87 118 24 214  (49-155)  (51-278) (8-72) (140-326) 2 2 1 1Phosphate 217 514 110  206 (132-355) (332-796) (66-183) (141-300) 2 2 10.5 Phosphate 105 381 90 206 (40-279) (180-808) (34-236)  (96-445) 2 2 12 Phosphate 155 374 53 502  (71-339) (196-713) (28-100) (335-752) 2 20.5 1 Phosphate 224 358 43 624 (125-400) (223-577) (14-128) (426-914) 22 2 1 Phosphate 180 306 70 423 (113-288) (190-492) (34-146) (258-696)

Serum bactericidal titres were as follows:

Antigen quantity (μg/dose) Aluminium Bactericidal antibody titre A CW135 Y adjuvant A C W135 Y 4 2 2 2 Phosphate 256-512 1024-2048 1024-20484096-8192 4 2 2 2 Hydroxide 1024-2048 256-512 1024-2048 1024-2048 2 2 22 Phosphate  512-1024 1024-2048 128-256  8192-16384 2 2 2 2 Hydroxide256 1024-2048 256  512-1024 4 2 1 1 Phosphate  512-1024 2048 1282048-4096 4 2 1 0.5 Phosphate  512-1024 1024-2048 128 2048-4096 4 2 1 2Phosphate  512-1024 2048-4096 128  8192-16384 4 2 0.5 1 Phosphate1024-2048 8192 256-512  8192-16384 4 2 2 1 Phosphate — 2048-4096 1284096-8192 2 2 1 1 Phosphate 1024-2048 1024-2048 256 4096-8192 2 2 1 0.5Phosphate 1024-2048 2048-4096 256-512 2048-4096 2 2 1 2 Phosphate 512-1024 1024-2048 128  8192-16384 2 2 0.5 1 Phosphate 1024-2048 2048256-512 4096-8192 2 2 2 1 Phosphate 128-256  512-1024  64-128 1024-2048

A second set of experiments was performed using a dosage of 2 μg/mlsaccharide for MenA and MenC, half that dosage for MenY, and a quarterdosage for MenW135. ELISA titres were as follows:

Antigen quantity (μg/dose) Aluminium GMT ELISA (95% confidence interval)A C W135 Y adjuvant A C W135 Y 2 2 2 2 Phosphate  32 114 99 397 (15-68) (53-246) (35-288) (252-627) Hydroxide 206 163 139  141 (112-372)(122-218) (76-251)  (97-205) 2 2 0.5 1 Phosphate  96 238 42 315 (49-187) (101-561) (20-89)  (114-867) Hydroxide 293 267 83 244(144-597) (158-451) (43-163) (152-392)

Serum bactericidal titres were as follows:

Antigen quantity (μg/dose) Aluminium A C W135 Y A C W Y adjuvant F8238A1 F6124 C11 5554 242317 242975 2 2 2 2 Phosphate 128-256 1024 1024-2048512 256-512 1024 2048 Hydroxide 512 1024-2048 1024-2048 512-1024 256-5121024 2048-4096 2 2 0.5 1 Phosphate 256 — 1024-2048 512 256-512 10242048-4096 Hydroxide 128 —  512-1024 512-1024  512-1024 1024 1024

L. MenA, W135 and Y Oligosaccharide Conjugates

The following table shows data relating to MenA, MenW135 and MenYconjugates suitable for making combination compositions of theinvention:

A W135 Y DP after sizing 16.6 21.9 21.1 Saccharide/protein ratio 0.5 1.10.7 KD 0.44 0.36 0.41 Free saccharide  5% 10%  5% Free protein <2% <2%<2%

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES The Contents of which are Hereby Incorporated in Full

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The invention claimed is:
 1. A method for conjugating a bacterialcapsular polysaccharide to a carrier protein, comprising (a) purifyingthe polysaccharide, comprising the steps of (i) precipitation of thepolysaccharide using one or more cationic detergents, followed by (ii)solubilisation of the precipitated polysaccharide using an alcohol, and(b) conjugating the polysaccharide to a carrier protein, wherein thebacterial capsular polysaccharide is from Neisseria meningitidisserogroup A, Neisseria meningitidis serogroup W135, Neisseriameningitidis serogroup Y, Haemophilus influenzae or Streptococcuspneumoniae, wherein the method further comprises a step of hydrolysis toform oligosaccharides prior to step (b).
 2. The method of claim 1,wherein the cationic detergent(s) have the following general formula:

wherein: R₁, R₂ and R₃ are the same or different and each signifiesalkyl or aryl; or R₁ and R₂ together with the nitrogen atom to whichthese are attached form a 5- or 6-membered saturated heterocyclic ring,and R₃ signifies alkyl or aryl; or R₁, R₂ and R₃ together with thenitrogen atom to which these are attached form a 5- or 6-memberheterocyclic ring, unsaturated at the nitrogen atom, R₄ signifies alkylor aryl, and X⁻ signifies an anion.
 3. The method of claim 1 or claim 2,wherein the one more cationic detergents comprise acetyltrimethylammonium salt, a tetrabutylammonium salt, amyristyltrimethylammonium salt or hexadimethrine bromide.
 4. The methodof claim 3, wherein the cationic detergent is cetyltrimethylammoniumbromide.
 5. The method of claim 1 or claim 2, wherein the alcohol usedin step (b) comprises ethanol.
 6. The method of claim 3, wherein thealcohol used in step (b) comprises ethanol.
 7. The method of claim 5,wherein the ethanol has a final concentration of between 50% and 95%. 8.The method of claim 6, wherein the ethanol has a final concentration ofbetween 50% and 95%.
 9. The method of claim 1 or claim 2, furthercomprising the step of (c) treating the polysaccharide obtained in step(b) to remove contaminants.
 10. The method of claim 6, furthercomprising the step of (c) treating the polysaccharide obtained in step(b) to remove contaminants.
 11. The method of claim 9, wherein step (c)comprises a step of filtration.
 12. The method of claim 10, wherein step(c) comprises a step of filtration.
 13. The method of claim 11, whereinthe filtration is one or more of depth filtration, filtration throughactivated carbon, size filtration and ultrafiltration.
 14. The method ofclaim 12, wherein the filtration is one or more of depth filtration,filtration through activated carbon, size filtration andultrafiltration.
 15. The method of claim 1 or claim 2, wherein thepolysaccharide obtained in step (b) or step (c) is then precipitated.16. The method of claim 10, wherein the polysaccharide obtained in step(b) or step (c) is then precipitated.
 17. The method of claim 1, furthercomprising the step of sizing in order to remove short-lengtholigosaccharides.
 18. The method of claim 10, further comprising thestep of sizing in order to remove short-length oligosaccharides.
 19. Themethod of claim 15, further comprising the step of sizing in order toremove short-length oligosaccharides.
 20. The method of claim 16,further comprising the step of sizing in order to remove short-lengtholigosaccharides.
 21. The method of claim 1, further comprising the stepof mixing with other biological molecules.
 22. The method of claim 21,wherein the further biological molecules are selected from the groupconsisting of: saccharide antigens from serogroup C of Neisseriameningitidis, and protein antigens from serogroup B of Neisseriameningitidis.
 23. The method of claim 21, wherein the other biologicalmolecules are one or more Neisseria meningitidis saccharide antigensfrom serogroup A, C, W135 or Y, or from a mixture thereof.
 24. Themethod of claim 1, further comprising the step(s) of vaccineformulation.
 25. The method of claim 24, wherein the step(s) of vaccineformulation comprises mixing the saccharide antigen(s) with an adjuvant.26. The method of claim 24, wherein the adjuvant is an aluminumphosphate adjuvant, an aluminum hydroxide adjuvant or a combinationthereof.
 27. A method for conjugating to a carrier protein bacterialcapsular polysaccharide that has been precipitated using one or morecationic detergents and then solubilized using ethanol as a solvent,wherein the capsular polysaccharide is from Neisseria meningitidisserogroup A, Neisseria meningitidis serogroup W135, Neisseriameningitidis serogroup Y, Haemophilus influenzae or Streptococcuspneumoniae, wherein the ethanol is in the form of at least 95:5ethanol:water.
 28. The method of claim 27, wherein the carrier proteinis selected from the group consisting of: a diphtheria toxoid, a tetanustoxoid and a CRM₁₉₇ diphtheria toxoid.
 29. The method of claim 27,wherein the ethanol is in the form of a 95:5 ethanol:water mixture.